This invention relates to antigenic P. falciparum merozoite surface polypeptides of approximate molecular weight 185,000 and processing fragments thereof. In particular, the invention relates to such polypeptides isolated by monoclonal antibody 5.2 of the invention. The polypeptides have use as vaccines.
Malaria is a serious health problem in many parts of the world. The disease is caused by a mosquito-borne parasite of the genus Plasmodium. Of greatest concern to humans are the Plasmodium falciparum and Plasmodium vivax species of the parasite.
The Plasmodium parasite has a complex life cycle. The parasite is introduced into the human body by the mosquito in the sporozoite form. The sporozoite travels to the human liver where it differentiates into the merozoite form of the parasite. Each merozoite, upon release from the liver, invades a red blood cell and goes through a series of stages (ring, trophozoite, schizont), eventually resulting in the formation and release of a large number (10-30) of merozoites. These merozoites then attack other red blood cells and the process continues unless arrested by medication or the body's immune system. As used herein, the term "asexual red blood cell stage parasite" shall be taken to embrace the various forms in which the parasite exists within the red blood cell, including merozoite, ring, trophozoite and schizont stages.
There have been a number of reports of work directed toward the possibility of developing a vaccine for either the sporozoite or merozoite stage of the malaria parasite. Work in the sporozoite area is disclosed in, e.g., U.S. Pat. No. 4,466,917 and UK Patent Application 2,145,092A (published Mar. 20, 1985).
Workers studying the merozoite in P. falciparum have reported a class of polypeptides in merozoites (and their schizont precursors) with molecular weights varying over the range of 185,000 to 200,000 (185K to 200K). The variation in reported molecular weights is in part a reflection of the fact that relative molecular weight (M.sub.r) estimates using SDS-PAGE are approximations, but the variation is also attributable to real differences in the polypeptides isolated from different malaria isolates. Molecules within this class of polypeptides have been referred to at various times, inter alia, as pf195, P195, P190, gp185, P200 and polymorphic schizont antigens (PSA). Polypeptides within this class have been reported on occasion to be surface molecules, to be antigenic in nature, and to be precursors for surface fragments of smaller molecular weight.
There have been a number of reports of work with monoclonal antibodies specific to polypeptides within the class of 185-200K P. falciparum merozoite polypeptides. L. H. Perrin et al., Clin. Exp. Immunol., 41, 91-96 (1980) reported immunoprecipitation of 195K 35-S-methionine protein (Senegal isolate) using monoclonal antibody 2B6. The protein did not inhibit parasite growth in vitro. A. A. Holder et al., J. Exp. Med., 156, 1528-1538 (1982) reported that monoclonal antibody 89.1 identified a 195K merozoite surface coat precursor protein (Wellcome-Lagos isolate). R. R. Freeman et al., J. Exp. Med., 158, 1647-1653 (1983) reported that the 195K precursor protein identified by monoclonal antibody 89.1 is processed to an 83K surface fragment. A. A. Holder et al., J. Exp. Med., 160, 624-629 (1984) reported that the 195K precursor protein identified by monoclonal antibody 89.1 gave rise to 42K and 19K surface fragments. R. J. Howard et al., Mol. Biochem. Parasit., 11, 349-362 (1984) reported the immunoprecipitation of a glycosylated 195K protein (St. Lucia isolate) with monoclonal antibodies PF27H10.19 and PF23H7.1. R. Hall et al., Mol. Biochem. Parasit., 11, 61-80 (1984) reported that monoclonal antibody 2.2 recognized a constant epitope while monoclonal antibody 7.3 recognized a variable epitope on a 190K merozoite surface protein (KI Thai isolate). P. H. Pirson et al., J. Immunol., 134, 1946-1951 (1985) reported that monoclonal antibody 5B1 specific to a processed 200K glycoprotein (Gambia isolate) partially inhibited parasite growth in vitro. J. McBride et al., J. Exp. Med., 161, 160-180 (1985), working primarily with a Thai isolate and a panel of strain-specific monoclonal antibodies, reported that P. falciparum consists of a number of antigenically diverse strains. They reported a "family" of polymorphic schizont antigens of M.sub.r 190-200K. R. Schmidt-Ullrich et al., J. Exp. Med., 163, 179-188 (1986), working with hybridomas KJ7-2C11D and KD8-2B2D, reported monoclonal antibodies binding to a 195K polypeptide (Gambia K1 isolate).
There have also been reports of attempts to demonstrate in vivo protection in immunization experiments using polypeptides from the class of merozoite 185-200K polypeptides. R. Hall et al., Nature, 31, 379-382 (1984) reported the results of an experiment in which three Saimiri monkeys were immunized with 190K polypeptide (from the Thai isolate K1 of P. falciparum) in Freund's complete adjuvant. The polypeptide used in the immunization was isolated using monoclonal antibody 7.3. Upon heterologous challenge with P. falciparum (Palo Alto strain), two of the monkeys developed parasitemias of from 5 to 10% before subsequently controlling the infection without drug treatment. The third monkey developed a parasitemia of greater than 20% and was drug treated. There was no increase in the prepatent period relative to the controls. At page 379 the authors described their work as follows: "Immunization with the affinity-purified native protein modifies the course of infection by the parasite."
In other work, L. H. Perrin et al., J. Exp. Med., 160, 441-451 (1984) reported the results of an experiment in which four Saimiri monkeys were immunized with polypeptides eluted from the 200K region of SDS-PAGE gels containing total P. falciparum (SGE2 Zaire isolate) polypeptides isolated by human sera. The polypeptides were not isolated or purified with monoclonal antibodies. Each monkey was immunized three times (once in Freud's complete adjuvant and twice in Freund's incomplete adjuvant) with polypeptides from the 200K region of the gel. Upon heterologous challenge with P. falciparum (Uganda-Palo Alto strain FUP), one monkey had a peak parasitemia of 11%; one of 6%; and two at a level described at page 445 as being "less than 3%" (the symbol for "less than" is used in the original). For each monkey the parasitemia eventually dropped off to a low level. In each monkey there was an increase in the prepatent period relative to the controls. See FIG. 3 of Perrin et al. at page 446.
The prior art shows the absence of, and the need for, a merozoite polypeptide which can be used in immunization to provide protection against P. falciparum parasitemias of the low levels associated with the symptoms of malaria. In humans the onset of fever and chills caused by malaria occurs at parasitemia levels of 0.5% or less; mortality can occur in non-immunes at levels of 2% parasitemia. Parasitemia is defined as the number of malaria-infected erythrocytes per 100 erythrocytes. It is measured by taking a blood sample from the infected animal or person, preparing stained slides, and reading the slides under the microscope.